Altered sulfation status of FAM20C-dependent chondroitin sulfate is associated with osteosclerotic bone dysplasia

Raine syndrome, a lethal osteosclerotic bone dysplasia in humans, is caused by loss-of-function mutations in FAM20C; however, Fam20c deficiency in mice does not recapitulate the human disorder, so the underlying pathoetiological mechanisms remain poorly understood. Here we show that FAM20C, in addition to the reported casein kinase activity, also fine-tunes the biosynthesis of chondroitin sulfate (CS) chains to impact bone homeostasis. Specifically, FAM20C with Raine-originated mutations loses the ability to interact with chondroitin 4-O-sulfotransferase-1, and is associated with reduced 4-sulfation/6-sulfation (4S/6S) ratio of CS chains and upregulated biomineralization in human osteosarcoma cells. By contrast, overexpressing chondroitin 6-O-sulfotransferase-1 reduces CS 4S/6S ratio, and induces osteoblast differentiation in vitro and higher bone mineral density in transgenic mice. Meanwhile, a potential xylose kinase activity of FAM20C does not impact CS 4S/6S ratio, and is not associated with Raine syndrome mutations. Our results thus implicate CS 4S/6S ratio imbalances caused by FAM20C mutations as a contributor of Raine syndrome etiology.

This is an interesting manuscript which reveals new insights into the role of FAM20C involvement in regulation of chondroitin sulfate (CS) biosynthesis, and genetic links with osteosclerotic bone dysplasia. The authors reveal for the first time that FAM20C has kinase activity for phosphorylation of the linkage tetrasaccharide, and also that is it interacts directly with the CS 4-O-sulfotransferase1 (4ST1) and can regulate the level of 4-sulfation and thus the 4S/6S ratio. Known genetic mutants in FAM20C were not associated with the Xyl-kinase activity, but were associated with lack of ability of the mutant proteins to interact with and enhance C4ST1 activity. The uthors contend that control of the 4S/6S ratio is crucial for physiological control of osteogenesis. Whilst they do provide a significant amount of data in Figs 4, 5 and part of Fig 6) that altering 6S biosynthesis to manipulate this ratio has effects on osteoblast differentiation and osteogenesis, this data is not a direct demonstration of the effects of altering 4ST1 activity through mutantions in FAM20C. The idea of FAM20C being critical for maintaining the correct sulfation balance between 4S and 6S of CS (and loss of this balance in FAM20C-related human genetic disorders) is certainly novel, but in its current form the manuscript does not providing convincing evidence.
For example it is puzzling that mutations in FAM20C reduce its interaction with 4STs, and decrease the 4S/6S ratio, whereas this shift in ratio increases osteoblast differentiation (which is the opposite of would be expected in the disease process. Similarly, increased 6S (through overexpression of C6ST) also decreases the 4S/6S ratio and also increases osteoblast differentiation, so this also seems counter to the authors arguments.
Clearly the system is quite complex, and subtle changes in 4S/6S ratios and the threshold levels may well be involved. In addition, there are noted differences between mouse and human systems. This makes it even more crucial that studies are undertaken on human cells to try to resolve these issues and make clarify how the results from mouse studies directly relevant to the human disease.

Major comments
The authors need to provide evidence of the effects of FAM20C mutations or at least FAM20C knockdown in relevant mouse cell lines, with analysis of altered 4S/6S ratios and concomitant alterations in osteogenesis-related outcomes which would be consistent with the disease phenotype. Although the extensive data provided on C6ST is supportive and interesting, it is insufficient to confirm the mechanisms underlying how mutant FAM20C functions.
Furthermore, the authors need to provide data on FAM20C overexpression and knockdown, and in human cells relevant to osteogenesis. Preferably this would also include examination of the activities of mutant FAM20C proteins.
Minor comments P3, changes in CS production (~2-fold) should be described as significant, rather than dramatic.
Can the authors confirm that the changes they claim to observe in CS chain length are greater than would be expected just from the increased level of sulfation (which will increase chain molecular weight, but not length).
Many spelling errors which need to be corrected in text and figures.

Reviewer #2
Expert in FAM20C (Remarks to the Author): Background: Fam20C is a member of a family of proteins that shared sequence similarity (Fam20A, B, C), but were of unknown function. Fam20 proteins also displayed weak similarity to a Drosophila protein kinase four-jointed (Fj) (1). It has previously been shown that Fam20C is a secretory pathway protein kinase that resides in the ER and Golgi, and can also be secreted outside cells (2,3). It has also been well-established that Fam20C is highly specific for serine residues within an SxE/pS motif found in a large number of secretory proteins (2)(3)(4). Mutations in Fam20C are causitive for Raine Syndrome, a severe and often lethal bone dysplasia characterized by osteosclerosis and ectopic calcifications (5). A second member of this family, Fam20B, has been shown to play an important role in controlling proteoglycan maturation of cartilage matrix proteins, chondrocyte maturation, and perichondral bone development in zebrafish (6). Fam20B has been shown to phosphorylate a xylose residue within the tetrasaccharide linkage region of O-linked proteoglycans, thereby regulating GAG chain maturation (7,8). The third member of this family, Fam20A, has recently been shown to allosterically potentiate Fam20C protein kinase activity, although Fam20A does not itself possess intrinsic kinase activity (9). Notably, previous studies using highly purified preparations of recombinant human Fam20C or the single ortholog present in C. elegans (ceFam20) have shown that Fam20C is highly specific for SxE/pS motifs within proteinaceous substrates and does not exhibit kinase activity toward artificial substrates that mimic xylose within the proteoglycan tetrasaccharide linkage (2,4,10). Likewise, highly purified recombinant Fam20B is active toward xylose within a minimal Gal-Xyl disaccharide an does not show activity toward SxE/pS motifs within proteinaceous substrates (7,8,10).
In the manuscript entitled, "Breakdown in FAM20C-mediated machinery for chondroitin sulfate biosynthesis causes osteosclerotic bone dysplasia", Koike et al presented evidence that the secretory kinase Fam20C functions to augment the 4S/6S sulfation ratio of proteoglycan chondroitin sulfate GAG chains. In this manuscript, the authors linked GAG abundance to Fam20C. In contrast to previous reports, they also showed that Fam20C may harbor intrinsic xylose kinase activity; however, this activity did not appear to be linked to Raine Syndrome. Overexpression of Fam20C in murine L cells increased the both the length and 4S/6S ratio of CS GAG chains. In contrast, these effects were not observed in a mutant cell line (sog9) that is deficient in the Golgi resident 4-O-sulfotransferase, C4ST-1. The authors used co-immunoprecipitation studies to provide evidence of a physical interaction between Fam20C and C4ST-1. They also observed an increase in relative 4-Osulfotransferase activity when Fam20C and C4ST-1 were co-expressed. The Fam20C/C4ST-1 interaction was abrogated by Fam20C mutations causitive for Raine Syndrome, suggesting that alterations in C4ST-1 activity and the 4S/6S ratio of CS GAG chains may play an important role in the etiology of Raine Syndrome patients. Finally, the authors employed studies with the murine osteoblast cell line, MC3T3-E1, and a transgenic mouse model in which overexpression of the 6-O-sulfotransferase, C6ST-1, was used to artificially decrease the 4S/6S CS ratio without dramatically lower the overall CS abundance. C6ST-1 overexpression decreased the 4S/6S CS ratio and accelerated MC3T3-E1 osteoblast differentiation. C6ST-1 overexpression in mice led to a decreased 4S/6S CS ratio with a concommitant increase in bone mineral density that is characteristic of Raine Syndrome patients.
The hypothesis that Fam20C may play a pivotal role in regulating proteoglycan CS GAG chain abundance and 4S/6S ratio is very intriguing. Furthermore, the findings of these studies might implicate loss-of-function Fam20C mutations in the development of osteosclerotic bone dysplasia associated with severe Raine Syndrome by an additional mechanism distinct from its protein kinase activity as reported previously. However, it is the opinion of this reviewer that there are a number of major concerns regarding the work and conclusions presented in this manuscript, as outlined below: 1) The demonstration that Fam20C harbors intrinsic xylose kinase activity is unconvincing in light of previous reports in the literature to the contrary using highly purified recombinant proteins. In addition, it has also been shown that the same Raine Syndrome mutations render Fam20C kinase inactive. More importantly, both severe and hypomorphic allele Fam20C mutant protein (SxE) kinase activities correlate directly with the severity of the phenotype, strongly suggesting that it is Fam20C protein kinase activity that is critical for disease progression. Further, mutant Fam20C was incapable of secretion outside the cell, which is in direct conflict with the data shown herein. The relative efficacy in knockdown experiments should also be shown by immunoblotting, preferably with more than a single oligo, to minimize the potential for off-target effects. The significant discrepancies between the current data and prior reports should be clarified. It has also been reported that Fam20C can physically interact with at least one other member of the family, Fam20A, suggesting that interactions between overexpressed Fam20C and other endogenous Fam20 kinases (activities) that might interfere with the clear interpretation of experimental data must be given serious consideration when conducting experiments of this kind. Given the possibility that the xylose kinase activity detected may have been an artifact caused by co-immunoprecipitation of some other secretory kinase present, it would have been more convincing had the authors used highly purified proteins, or demonstrated by immunoblotting that endogenous Fam20A/B/C were present only where expected in the samples used for xylose kinase activity analyses. Alternatively, the authors could have imunoprecipitated proteins used in kinase assays from Fam20B-deficient cells. Further, the authors demonstrate that Fam20C xylose kinase activity was not affected by Raine Syndrome mutations and suggest that it likely has no role in CS GAG chain elongation or modulating 4S/6S ratio. It is this reviewer's opinion that the early focus on Fam20C xylose kinase activity was a detraction from the story as a whole, representing primarily negative data.
2) As mentioned above, previous reports have demonstrated that Fam20C is a Golgi protein kinase that phosphorylates a wide range of secretory pathway proteins within a highly conserved SxE/pS motif. Surprisingly, the authors did not test the possibility of Fam20C might directly phosphorylate C4ST-1, which in turn could affect its transferase activity. C4ST-1 contains a well-conserved SxE motif within its C-terminus that could conceivably serve as a Fam20C target. It is unclear whether overexpression/knockdown of Fam20C could affect the expression/secretion level of C4ST-1 or other glycosyltransferases, as has been previously observed.
3) The authors should more thoroughly quantify and characterize the xylose kinase substrate, ITI. Clearly is difficult to generate these glycosylated peptides, but it is not acceptable for the authors to just assume they will get the correct glycosylation when they mixed the "bead-pulled glycosyltransferase" and UDP-sugar (pg 12). In addition, the second step of the reaction involves phosphorylation of the "xylosylated peptide (Xyl-o-TIT) by Fam20B. Previous work including the authors themselves has demonstrated that Fam20B cannot efficiently use Xyl-O-Ser as a substrate, if at all. This discrepancy should be addressed.
4) The experiments used by the authors to draw the conclusion that FAM20C can regulate GAG abundance was not convincing. Specifically, the amount of GAG was normalized to dry weight. It is unclear whether this normalization makes sense considering the possible effect of Fam20C on protein secretion (Fig 1a, 1b). Also, the relationship between GAG chain length and 4S/6S ratio were not clearly explained. Although changes in both were observed, further explanation as to whether those processes are interrelated would be of value. Both CS and HS GAGs were affected by Fam20C overexpression based on the authors' observations, but they did not further explain or comment on the effects as related to HS chains (Fig 1a, 1b, 1c, 1d). In addition, the observed effects were only statistically significant when Fam20C was overexpressed ( Fig 1E). The effect of Fam20C silencing was not statistically different than the control, raising the question of physiological relevance. Finally, the change in C4 sulfation was previously reported in the authors' Fam20B paper, where they showed that Fam20B overexpression could increase C4 sulfation and Fam20B knockdown decreased C4 sulfation. This seems to be at odds with their current findings and should be discussed further. Fig 2b, it appears as though the authors radiolabeled the cells with 32P, then treated the cells with base to release O-linked sugars. How do they know they are measuring Gal-Gal-Xyl(P) or Gal-Xyl(P)? It would be more convincing if the authors had structurally characterized what they actually measured, because base treatment can release many sugars, some that may also contain phosphate. Second, a better method of normalization or internal control would be more convincing than % of total radioactivity.

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6) The co-immunoprecipitation between C4ST-1 and Fam20C was unconvincing as both Fam20C and C4ST were overepressed in the cells. Co-IP might be an artifact. It would have been more convincing had at least one protein been endogenous. In addition, previous reports have indicated that the same Fam20C Raine Syndrome mutants were incapable of being secreted outside the cell. It is unclear how the authors were able to isolate these proteins from conditioned medium for use in enzymatic assays. This discrepancy should be discussed further. 7) Based on the hypothesized relationship between Fam20C and CS4T-1, the authors utilized systems in which they artificially manipulated levels of C6ST-1 to lower the 4S/6S ratio without significantly altering overall CS production. Although alteration of the 4S/6S ratio by these means clearly affected bone mineralization, it is unclear whether this might also occur by a mechanism independent of Fam20C/C4ST-1. Is it possible through these experiments to conclude that the changes initially observed in the 4S/6S ratio caused by Fam20C overexpression weren't affected by shortening of CS chains, which also was observed? -

Reviewer #3
Expert in osteoblastogenesis (Remarks to the Author): In the manuscript "Breakdown in FAM20C mediated machinery for chondroitin sulfate biosynthesis causes osteosclerostic bone dysplasia" the authors evaluated FAM20C functions and the effect of common Raine syndrome mutations in the FAM20C locus to and provide new insights into the pathogenic mechanisms of osteosclerostic bone dysplasia.
Even though the topic is very complex, they provided a very concise and comprehensible report on the probable mechanism of FAM20C.
I have the following comments: 1. The authors should provide information on the statistics for all figures.
2. Readers unfamiliar with the process of GAG synthesis would benefit from the introduction of a brief figure on the linkage tetrasaccharide synthesis and chain elongation process early in the manuscript. Also, key references on GAG sulfation are missing.
3. Please provide a reference for the statement in the discussion that the 4S/6S ratio in mice differs from humans 4) Do FAM20 mutations only affect osteoblasts or other bone cell types (osteoclasts, osteocytes) as well? How are the bone turnover marker in affected humans? Alterations in GAG sulfation are known to directly affect bone cells and signaling molecules.
Response to the Referee's Comments 1 We are deeply grateful for their careful review and constructive comments on our manuscript.  Can the authors confirm that the changes they claim to observe in CS chain length are greater 2 than would be expected just from the increased level of sulfation (which will increase chain 3 molecular weight, but not length).  Background: Fam20C is a member of a family of proteins that shared sequence similarity 2 (Fam20A, B, C), but were of unknown function. Fam20 proteins also displayed weak 3 similarity to a Drosophila protein kinase four-jointed (Fj) (1). It has previously been shown that 4 Fam20C is a secretory pathway protein kinase that resides in the ER and Golgi, and can also be 5 secreted outside cells (2,3). It has also been well-established that Fam20C is highly specific 6 for serine residues within an SxE/pS motif found in a large number of secretory proteins (2-4).  Table 2). The associated comments were added in the text (lines 109-113). 31 4) Although XYLK activity-dependent regulation of GAG abundance is not correlated with 1 Raine syndrome, the data for the substrate preferences of FAM20C mutants as a XYLK 2 provide an important insight into the FAM20B-like functional properties of FAM20C 3 mutants. Therefore, the XYLK-related data remain to be presented in the revised manuscript 4 (Fig. 2, Supplementary Figs. 2-3, and Supplementary Tables 2-3), and the associated 5 comments were added in the text (lines 156-158, and 280-288). 3) The authors should more thoroughly quantify and characterize the xylose kinase substrate, 23 ITI. Clearly is difficult to generate these glycosylated peptides, but it is not acceptable for the 24 authors to just assume they will get the correct glycosylation when they mixed the "bead-pulled

31
According to the reviewer's comment, we attempted to characterize the ITI-substrates. However, 32 since the yields of the respective ITI-substrates appeared to be quite low, complete structural analysis of ITI-substrates was so tough. Despite these uncertainties, apparent substrate 1 preferences of FAM20 kinases were clearly detected using the ITI-substrates; indeed, 2 Xyl1-O-ITI was a less effective substrate for both FAM20B and FAM20C, as reported 3 previously (Ref. 27 in the revised manuscript). Therefore, we believe the usefulness of 4 ITI-substrate for sensitive detection of Xyl kinase activity.

6
4) The experiments used by the authors to draw the conclusion that FAM20C can regulate 7 GAG abundance was not convincing. Specifically, the amount of GAG was normalized to dry 8 weight. It is unclear whether this normalization makes sense considering the possible effect of 9 Fam20C on protein secretion (Fig 1a, 1b). Also, the relationship between GAG chain length 10 and 4S/6S ratio were not clearly explained. Although changes in both were observed, further 11 explanation as to whether those processes are interrelated would be of value.

12
Both CS and HS GAGs were affected by Fam20C overexpression based on the authors' 13 observations, but they did not further explain or comment on the effects as related to HS chains 14 (Fig 1a, 1b, 1c, 1d). In addition, the observed effects were only statistically significant when 15 Fam20C was overexpressed (Fig 1E). The effect of Fam20C silencing was not statistically 16 different than the control, raising the question of physiological relevance. Finally, the change 17 in C4 sulfation was previously reported in the authors' Fam20B paper, where they showed that 18 Fam20B overexpression could increase C4 sulfation and Fam20B knockdown decreased C4 19 sulfation. This seems to be at odds with their current findings and should be discussed further.  Therefore, we believe that such a slight decrease in 4S/6S ratio is also a physiologically 8 important change. intermediates on a Superdex peptide gel filtration column. Taking this advantage, we also 31 measured the radioactivity of the corresponding peaks derived from the respective HeLa cells manipulating expression of FAM20 proteins in this study. That is why the ratios of the 1 radioactivity of two major intermediates were shown in Fig. 2b (in the original manuscript).

2
According to the reviewer's comment, we modified Fig. 2b, and